Dynamin is an ~100 kD multidomain GTPase required for clathrin-mediated endocytosis, but its exact role remains unknown. It self-assembles into rings and helical stacks of tings and self-assembly stimulates dynamin's GTPase activity -100-fold. In the presence of GTPgammaS, dynamin spirals (called 'collars') accumulate around the necks of deeply invaginated coated pits. Endocytosis is inhibited by overexpression of dynamin mutants defective in GTP binding and/or hydrolysis. Based on these observations, we first proposed a model for dynamin function as a mechanochemical 'pinchase', in which GTP hydrolysis drives a concerted conformational change to constrict the assembled dynamin collar and drive membrane fission. In probing the mechanisms that coordinate self-assembly with stimulated GTP hydrolysis we discovered that the GTPase effector domain, or GED, in dynamin is required for self-assembly and functions as an assembly-dependent GAP. Surprisingly, endocytosis was stimulated in cells overexpressing dynamin with a mutation in GED that impairs self-assembly and consequently assembly-dependent GAP activity. This unexpected result led us to propose an alternate model suggesting that dynamin functions (like all other GTPase superfamily members) as a regulatory enzyme controlling downstream effectors required for vesicle formation. Recent evidence that dynamin expression can alter actin dynamics, affect cell shape changes and trigger a signaling cascade leading the p53 activation and apoptosis is consistent with a broader role for dynamin in cellular signaling. However, considerable controversy exists as to the exact function of dynamin in clathrin-mediated endocytosis. Fundamentally, the critical question becomes: Is dynamin the 'brains' or the 'brawns' behind CCV formation? We propose to answer this question by pursuing the following Specific Aims: 1) Analyze the biochemical properties of the human homologues of the shibire-ts2 allele and 3 intra-allelic suppressors of shibire (sushi) 2) Generate and phenotypically characterize dynamin-2 knock-out cells and possibly mice 3) Utilize fluorescence-based microscopic assays to monitor dynamin's effects on the hierarchy of events leading to coated pit maturation and coated vesicle formation. 4) Utilize a newly developed assay for CCV formation from isolated plasma membranes to dissect dynamin function in vitro 5) Identify downstream effector molecules required for dynamin function in the cell.